1. Volume 112, Issue 1, Pages 87-98 (January 2017)
Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels 
Hermanus Johannes Ruigrok, Guillaume Shahid, Bertrand Goudeau, Florence Poulletier de Gannes, Emmanuelle Poque-Haro, Annabelle Hurtier, Isabelle Lagroye, Pierre Vacher, Stéphane Arbault, Neso Sojic, Bernard Veyret, Yann Percherancier 
Biophysical Journal 
Volume 112, Issue 1, Pages (January 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
Measuring TRPV1 activity using an intramolecular BRET probe (A). Schematic representation of the YFP-TRPV1-Luc intramolecular BRET probe, where YFP was fused to the N-terminal extremity of TRPV1 while Luc was fused to the C-terminal extremity of TRPV1. After activation of TRPV1, the distance (d) and/or the orientation (o) between Luc and YFP were expected to be modified. (B) Kinetic measurement of the effect of 20 μM CAPS on HEK293T cells expressing the YFP-TRPV1-Luc BRET probe. (Star) Time of CAPS injection. Data represent one out of five independent experiments. The time-constant (τ) of the BRET increase induced by CAPS is 2.01 ± 0.29 s, n = 5. To see this figure in color, go online.
Biophysical Journal  , 87-98DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. Figure 2
Measuring TRPV1 activity using an intermolecular BRET probe. (A) Schematic representation of the intermolecular BRET between TRPV1 and Calmodulin. YFP was fused to the N-terminal of Calmodulin and Luc to the C-terminal of TRPV1. After activation of TRPV1, the distance d between Luc and YFP was expected to be modified. (B) Kinetic measurement of the effect of 20 μM CAPS on cells expressing TRPV1-Luc and either YFP-CaM WT or YFP-CaM1234, pretreated or not with 10 μM BAPTA-AM for 30 min before activation with CAPS. (Star) Time of CAPS injection. Data represent one out of three independent experiments. (C) Titration assays using HEK293T cells transfected with increasing amounts of YFP-CaM WT (circles), YFP-CaM1234 (diamonds), or unfused YFP (square) and a fixed amount of TRPV1-Luc. Transfected cells were activated (open symbols) or not (solid symbols) with 20 μM CAPS before BRET reading. Results represent the data obtained over three independent experiments performed in duplicate. To see this figure in color, go online.
Biophysical Journal  , 87-98DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

4. Figure 3
Dose-response curves of CAPS-induced BRET changes measured in HEK293T cells expressing the YFP-TRPV1-Luc BRET probe (A) or the TRPV1-Luc/YFP-CaM constructs (B). Before CAPS activation, the cells were preincubated with either PBS, DMSO (vehicle), 1 μM CPZ, or 1 μM AMG517. In the presence of CPZ or AMG517, but not in the presence of the vehicle alone, the dose-response curve shifted to higher values and the EC50 was displaced by more than one order of magnitude (see also Table S1). Results represent the mean ± SE of four independent experiments done in duplicate.
Biophysical Journal  , 87-98DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

5. Figure 4
Effect of temperature on the pharmacological properties of TRPV1 and its activation, measured by BRET. (A and B) BRET ratios of HEK293T cells expressing either the TRPV1-Luc/YFP-CaM constructs (A) or the YFP-TRPV1-Luc BRET probe (B) were recorded in real time while the cell-culture medium was heated from 25 to 50°C using a Peltier plate. The effects of calcium-free extracellular medium (A) or preincubation of cells with 1 μM CPZ (A and B), were also tested. BRET ratios were corrected for the minor variation in Luc and YFP emissions due to the temperature increase (see Materials and Methods). In (A), the fact that the signal remained stable over the whole temperature range under Calcium-depleted conditions and in the presence of CPZ further excluded any bias in BRET measurements due to the denaturation of the Luc or YFP groups. Data represent one out of three independent experiments. (C and D) CAPS dose response curves measured by BRET using HEK293T cells, expressing either the TRPV1-Luc/YFP-CaM constructs (C) or the YFP-TRPV1-Luc BRET probe (D), incubated at 25, 31, 37, or 42°C. Results represent the mean ± SE of four independent experiments done in duplicate and are expressed as the difference between the net BRET measured in each condition and the basal BRET measured without any agonist at 37°C.
Biophysical Journal  , 87-98DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

6. Figure 5
Dose-response curve of the effect of Drofenine (A), CAPS (B), and GSK A (C) on HEK293T cells coexpressing YFP-CaM and Luc-TRPV3 (triangle), TRPV1-Luc (square), or TRPV4-Luc (circle). Results are expressed as the difference between the net BRET measured in each condition and the basal BRET measured without any agonist. Basal BRETs of ± 0.004, ± 0.003, and ± were measured in resting HEK293T cells expressing Luc-TRPV3/YFP-CaM, TRPV1-Luc/YFP-CaM, and TRPV4-Luc/YFP-CaM, respectively. As mentioned for TRPV1, these results potentially indicate that TRPV3 and TRPV4 interacted with CaM under nonactivated conditions. Results represent the mean ± SE of three independent experiments done in duplicate. To see this figure in color, go online.
Biophysical Journal  , 87-98DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

7. Figure 6
Multiplexing measurements of TRPV activity using multicolor BRET. (A) Example of a three-color BRET spectrum and its decomposition, measured in a coculture containing three HEK293T subpopulations transfected with aquamarine-Luc, mAmetrine-Luc, or LSSmOrange-Luc. (Gray dots) Experimental data. (Red line) Best fit to the experimental data. (Purple, blue, green, and orange lines) Spectral components of Luc, aquamarine, mAmetrine, and LSSmOrange, respectively. (B–D) Multicolored BRET produced by Luc and aquamarine, Luc and mAmetrine, and Luc and LSSmOrange were measured in real time in one sample containing a mixed population of cells expressing Luc-TRPV3/aquamarine-CaM (blue line, basal BRET of ± 0.002), TRPV1-Luc/mAmetrine-CaM (green line, basal BRET of ± 0.001), and TRPV4-Luc/LSSmOrange-CaM (red line, basal BRET of ± 0.001) constructs. One mM Drofenine (B), 20 μM CAPS (C), or 100 nM GSK101 (D) were injected 75 s after the beginning of the experiment initiated by the injection of purple coelenterazine into the buffer. Results represent the mean of three independent experiments.
Biophysical Journal  , 87-98DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions